Antibodies specifically bind to substances called antigens, and detoxify and remove antigen-containing factors with the cooperation of other biomolecules and cells. The name “antibody” is particularly based on such an antigen-binding ability, and is also referred to as “immunoglobulin (Ig)” as a chemical name.
Recent developments in genetic engineering, protein engineering, and cell technology have accelerated the development of antibody drugs, which are pharmaceuticals utilizing the abilities of antibodies. Since the antibody drugs more specifically attack a target molecule than conventional pharmaceuticals, use thereof is expected to further reduce side effects and to produce higher therapeutic effects. In fact, these drugs contribute to improvement in various disease conditions.
The quality of antibody drugs is thought to largely depend on the purity compared with the quality of other recombinant protein pharmaceuticals because the doses of these antibody drugs to the body are very large. In order to produce a high purity antibody, techniques using an adsorbing material that contains a ligand molecule capable of specifically binding to an antibody (e.g. affinity chromatography) are commonly employed.
Antibody drugs developed so far are generally monoclonal antibodies. These antibodies are mass produced by recombinant cell-culture technology or the like. The “monoclonal antibodies” refer to antibodies produced by clones of a single antibody-producing cell. Almost all antibody drugs currently available on the market are classified into immunoglobulin G (IgG) subclasses based on their molecular structures. One well-known example of immunoglobulin-binding proteins having affinities for IgG antibodies is Protein A. Protein A is a cell wall protein produced by the gram-positive bacterium Staphylococcus aureus and contains a signal sequence S, five immunoglobulin-binding domains (E domain, D domain, A domain, B domain and C domain) and a cell wall-anchoring domain known as XM region (Non Patent Literature 1). In the initial purification step (capturing step) in the process of antibody drug manufacture, affinity chromatography columns where Protein A is immobilized as a ligand on a water-insoluble carrier (hereinafter, referred to as Protein A columns) are commonly used (Non Patent Literatures 1 to 3).
Various techniques for improving the performance of Protein A columns have been developed. Various technological developments in ligands have also been made. Initially, wild-type Protein A has been used as a ligand, but currently, recombinant Protein A altered by protein engineering is used as a ligand in many techniques for improving the column performance.
Typical examples of such recombinant Protein A include recombinant Protein A without the XM region that does not bind to immunoglobulins (rProtein A Sepharose (registered trademark) available from GE health care, Japan). Currently, columns containing as a ligand recombinant Protein A without the XM region are widely used for industrial purposes because these columns have an advantage of suppressing non-specific adsorption of proteins compared with conventional ones.
Also known are inventive techniques in which a recombinant Protein A obtained by introducing a single Cys mutation into Protein A (Patent Literature 1) or a recombinant Protein A obtained by introducing a plurality of Lys mutations (Patent Literature 2) is used as a ligand. These techniques are effective in immobilization of ligands on a water-insoluble carrier and are advantageous in terms of the antibody-binding capacity of columns and for reducing leakage of the immobilized ligands.
Another well-known technique is using, as an engineered recombinant Protein A ligand, an engineered domain obtained by introducing mutation into the B domain (this engineered domain is referred to as Z domain) (Non Patent Literatures 1 and 4 and Patent Literature 3). Specifically, the Z domain is an engineered domain containing a substitution of Ala for Gly at position 29 of the B domain. In the Z domain, a substitution of Val for Ala at position 1 of the B domain is also contained, and this mutation is intended to facilitate genetic engineering preparation of a gene encoding multiple connected domains and does not affect the domain functions (for example, a variant containing a substitution of Ala for Val at position 1 of the Z domain is used in an example of Patent Literature 4).
The Z domain is known to be more alkali resistant than the B domain and has an advantage in reuse of a column through washing with an alkaline solution having high bactericidal and cleansing effect. Patent Literatures 5 and 6 disclose inventive ligands derived from the Z domain, containing a substitution of another amino acid for Asn in order to impart higher alkali resistance, and these ligands are already used for industrial purposes.
As described above, it is widely known that the substitution of Ala for Gly at position 29 of a immunoglobulin-binding domain (E, D, A, B or C domain) of Protein A is a useful mutation strategy. In fact, the prior Protein A engineering technologies developed after the disclosure of the “G29A” mutation in 1987 involve the “G29A” mutation (Patent Literatures 2, 4 and 6).
Another feature of the Z domain is its reduced binding ability to the Fab region of immunoglobulins (Non Patent Literature 5). This feature advantageously facilitates dissociation of an antibody binding to the domain with the use of an acid (Non Patent Literature 1 and Patent Literature 7). If an antibody readily dissociates, an eluate having a higher concentration of antibodies can be recovered using less eluant. Recent developments in antibody drug manufacture have increased the cell culture production capacity beyond 10,000 liters per batch, and in the past few years the antibody expression level has been improved up to nearly 10 g/L (Non Patent Literature 6). These developments have naturally created a need for scale-up of the processing capacity of the downstream purification process, and there is a very large demand for technical improvement in order to recover an eluate having a higher concentration of antibodies by using less eluant.
In addition to the Z domain, engineered Protein A ligands derived from the C domain of Protein A have also been studied (Patent Literature 4). These ligands characteristically take advantage of the inherent high alkali resistance of the wild-type C domain and have been receiving attentions as new alternative base domains to the Z domain prepared based on the B domain. However, our studies on the C domain have revealed a disadvantage that it is difficult to dissociate an antibody binding to the C domain with the use of an acid. The C domain, as taught in Non Patent Literature 2 and Patent Literature 4, has a strong binding ability to the Fab region of immunoglobulins, and this ability is presumed to make it difficult to dissociate the antibody with an acid. In order to overcome this disadvantage, we have examined a C domain variant containing a substitution of Ala for Gly at position 29 for its antibody dissociation properties in the acidic condition. The results have revealed that the antibody tends to more readily dissociate from the domain variant than the wild-type C domain, but its properties are not enough yet.
At present, the “G29A” mutation is the only one mutation which is known to cause an antibody to readily dissociate from the immunoglobulin-binding domains of Protein A, as described above. The “G29A” mutation has the above-mentioned advantages as well as ready dissociation of an antibody, and further technical improvement by mutations at positions other than position 29 is demanded. However, there are so far no reports of mutations at positions other than position 29 which enable readier dissociation of an antibody.